tim aitman [email protected] genome resources and identification of complex trait genes...
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Genome Resources and Identification of Complex Trait Genes
Physiological Genomics and MedicineMRC Clinical Sciences CentreFaculty of Medicine, Imperial CollegeHammersmith HospitalLondon
Identification of Genes underlying Mendelian and Complex Traits
1980-2002
Glazier, Nadeau, Aitman, 2002
No. of Mendelian traits
No. of Complex Traits
Mendelian traits
All complex traits
Human complex traits
1980 1985 1990 1995 2000
Genome Resources for Identifying Complex Trait Genes
• Genome sequences- Annotation, homology searching, comparative mapping, SNP resources, text mining
• Omics technologies- Transcriptional arraying, proteomics
• Congenic, isogenic, near isogenic strains-Rodents, plants, flies
• LD and haplotype maps- Humans, rodents
Criteria for Establishing Proof of QTL Gene Identity in Whole-Genome Linkage Studies
• Coarse mapping with genome-wide significance• Fine mapping
– Congenic strains, advanced intercrosses– LD mapping
• Sequence analysis• Functional testing of proposed sequence
nucleotide variant(s)– In vivo or in vitro complementation
• Circumstantial evidence – Formal proof vs. "wealth of evidence"
InsulinResistance
Beta cell failure
Diabetes
Salt retention
Hypertension
Hepatic lipoprotein overproduction
Dyslipidaemia
The Human Insulin Resistance Syndromes(Metabolic Syndrome X)
Predominant causes of coronary heart disease
THE SPONTANEOUSLY HYPERTENSIVE RAT:A model of the common insulin resistance syndromes
• Spontaneous hypertension
• Decreased insulin action
• Hyperinsulinaemia
• Central obesity
• Defective fatty acid metabolism
• Hypertriglyceridaemia
Intermediate phenotype: In vitro insulin and catecholamine resistance in SHR fat cells
Identification of Cd36 as Rat Insulin Resistance Gene
Combined DNA Microarrays and Linkage Analysis to Investigate Insulin Resistance in Hypertensive Rats
QTL Plots of Chromosome 4 for Defects in Insulin Action and Fatty
Acid Metabolism
0
2
4
6
8Lod
0
1
2
3
4
Wox21Ae2
Arb13Il6
Wox7Mgh4
Mgh17 Mgh8
10 cM
Ae2Arb13Il6
Wox7Wox21Mgh4
Mgh17 Mgh8
10 cM
+
Microarray to Detect Differential Gene Expression between Tissues from
Affected and Control Animals
Aitman et al, Nature Genet 1997 Aitman et al, Nature Genet 1999
F2 cross Backcross
Chromosome 4 Congenic Strain Confirms Linkage and Defines Physical Limit of QTL -
to 36 cM (= 72 Mbp)__________________________________________________________________________________________
Fatty Acid Secretion
BN SHR SHR.4
Glucose Uptake
BN SHR SHR.4
P = 0.0002 P = 0.01 P = 0.003 P = 0.009
Radiation Hybrid Mapping Places Cd36 in the Centre of the Chromosome 4 QTL
Asn102SerAla145Val
Thr150Gln
Val154Phe
Glu240LysAla401Pro
Ser468Phe
Lys183Glu
Ser160IleLeu161Phe
Val190Ile
5’ 3’
G397A G601A
G791A
C871T C1339T T1450C
C619T
"Exon 6"
cDNA Sequencing Identifies Multiple Mutations in SHR Cd36 Coding Region
“Exon 6”
Western Blot Analysis: Cd36 Protein is Undetectable in Plasma Membrane/Microsomes
from SHR Adipose Tissue
Chromosomal Deletion at Cd36 Locus Underlies Cd36 Deficiency in SHR
______________________________________________________________________________________________________________________________________________________
Cd36 Cd36-ps1
Chimaeric Cd36 in SHR
I II III IV V VI VII VIII IX X XI XII XIII XIV XV
I
Cd36-ps2
II III IV V VI VII VIII IX X XI XII XIII XIV XV II III IV V VI VII VIII IX X XI XII XIII XIV XVII II III IV V VI VII VIII IX X XI XII XIII XIV XV
: Promoter
Normal rat Cd36 locus
Glazier et al, Mamm Genome, 2001
How do you prove that a candidate gene, at the peak of linkage and with
strong biological candidacy, underlies a QTL?
“Proof” that Cd36 underlies SHR QTLs
• Cd36 transgenic mouse (Aitman 1999)• Cd36 knockout mouse (Febbraio 1999)• Human Cd36 deficiency (Miyaoka 2001)• Cd36 transgenic SHR (Pravenec 2001)
“Proof” that Cd36 underlies SHR QTLs
• Cd36 transgenic mouse (Aitman 1999)• Cd36 knockout mouse (Febbraio 1999)• Human Cd36 deficiency (Miyaoka 2001)• Cd36 transgenic SHR (Pravenec 2001)
PLASMA LIPIDS IN Cd36 KNOCKOUT MICE
Triglycerides NEFA (mg/dl)
(mEq/l) _______________________
Control 97.3 ± 5.9* 1.73 ± 0.09*
Knockout 138 ± 10.0 3.28 ± 0.12
__________________________________________* P<0.005
Febbraio et al, 1999J Biol Chem 274:19055
CLINICAL PROFILES OF HUMAN CD36 DEFICIENCY
_____________________________________________
CD36 Deficiency Control
(N=26) (N=96)Age (years) 64 ± 10 60 ± 7
Sex (Male, Female) (15,11) (68,28)
BMI (kg/m²) 23.6 ± 3.8 23.6 ± 2.7
TC (mmol/l) 5.29 ± 1.01 5.3 ± 0.83
TG (mmol/l) 2.19 ± 1.15 * 1.42 ± 0.7
HDL-C (mmol/l) 1.24 ± 0.42 * 1.61 ± 0.42
FPG (mmol/1) 6.18 ± 1.24 * 5.44 ± 1.11
Systolic BP (mmHg) 135 ± 19 * 118 ± 15
Whole Body Glucose Uptake 5.08 ± 1.4 * 8.6 ± 0.5
_______________________________________________________________* p<0.01 Miyaoka et al 2001, Lancet 357:686
CORRECTION OF METABOLIC AND CARDIOVASCULAR PHENOTYPES IN Cd36
TRANSGENIC SHR ______________________________________________________________________________
TG10 TG19 SHR
Plasma NEFA (mM) 0.30±0.01* 0.28 ±0.03* 0.44 ±0.04
Glucose AUC (mM.hr) 12.4 ±0.8* 11.6 ±0.4* 15.5 ±1.10
Glucose uptake (mM/g) 226 ± 16* 341 ±95* 143 ± 15_____________________________________________________________Values are means ± se; *P<0.05
Pravenec et al, 2001Nature Genet 27:156
Lines of evidence supporting Cd36 as SHR QTL gene
• Cd36 lies at peak of linkage • Linkage confirmed in congenic strains• Strong biological candidacy• Altered genomic locus with multiple coding
sequence variants• Undetectable protein on Western blot• Comparable phenotypes in Cd36-deficient mice
and humans• Transgenic complementation
How successful is the combined linkage and microarray approach?
Successful combined use of linkage analysis and microarray-based expression
profiling
• Complex traits– Insulin resistance (Aitman et al 1999)– Experimental asthma (Karp 2000)
• Mendelian traits– Sitosterolemia (Berge 2000)– Tangier disease (Lawn 1999)
Future combined use of microarrays and genetic linkage analysis to define
genetics of gene expression
• Genetical genomics– Jansen & Nap 2001
• Budding yeast – Brem et al 2002
• Maize, mouse and man– Schadt et al 2003– 11,021 eQTLs (of 23,574 genes on array)
with Lod > 3
Current Gene Identification Projects
• Salt-sensitive hypertension in SHR
• Pulmonary hypertension in F344 rats
• Nephrotoxic nephritis in WKY rat
1% salt solutionfor drinking
1% salt solutionfor drinking
100
110
120
130
140
150
80 90 100 110 120(days of age)
mm
Hg
(days of age)
150
160
170
180
190
90 100 110 12080
mm
Hg
Night-time, diastolic160
100
110
120
130
140
150
80 90 100 110 120(days of age)
1% salt solutionfor drinking
mm
Hg
150
160
170
180
190
200
210
80
mm
Hg
90 100 110 120(days of age)
1% salt solutionfor drinking
Night-time, systolic
Day-time, systolicDay-time, diastolic
SHR
SHR.18
24 Hour Blood Pressure Measured by Telemetry in SHR and SHR.18 Congenic Strain
SHR
SHR.18
SHR
SHR.18
SHR
SHR.18
D18Rat32
Ttr
D18Rat12Adrb
10cM*
Human BP QTL (Krushkal et al, 1999)Mouse BP QTL (Wright et al, 1999)*
Ga
rre
tt e
t a
l 1
99
8,
Da
hl
S/L
ew
Ja
co
b e
t a
l 1
99
1,
SH
RS
P/W
KY
Co
wle
y e
t a
l 2
00
0,
BN
/Da
hl
S
Ko
va
cs
et
al
19
97
, S
HR
/BB
OK
Previously Mapped Rat BP QTLs (95% C.l.)
S
HR
.18
Co
ng
enic
in
terv
al
Rat Chr. 18
Differentially Expressed Genes and BP QTLs on Rat Chromsome 18
D18Rat32DE4
Ttr
D18Rat12Adrb
DE1
DE2
DE3
GeneticMarkers
Differentiallyexpressed genes
10cM*
Human BP QTL (Krushkal et al, 1999)Mouse BP QTL (Wright et al, 1999)*
Ga
rre
tt e
t a
l 1
99
8,
Da
hl
S/L
ew
Ja
co
b e
t a
l 1
99
1,
SH
RS
P/W
KY
Co
wle
y e
t a
l 2
00
0,
BN
/Da
hl
S
Ko
va
cs
et
al
19
97
, S
HR
/BB
OK
Previously Mapped Rat BP QTLs (95% C.l.)
S
HR
.18
Co
ng
enic
in
terv
al
Rat Chr. 18
Differentially Expressed Genes and BP QTLs on Rat Chromsome 18
D18Rat32DE4
Ttr
D18Rat12Adrb
DE1
DE2
DE3
GeneticMarkers
Differentiallyexpressed genes
10cM*
Human BP QTL (Krushkal et al, 1999)Mouse BP QTL (Wright et al, 1999)*
Ga
rre
tt e
t a
l 1
99
8,
Da
hl
S/L
ew
Ja
co
b e
t a
l 1
99
1,
SH
RS
P/W
KY
Co
wle
y e
t a
l 2
00
0,
BN
/Da
hl
S
Ko
va
cs
et
al
19
97
, S
HR
/BB
OK
Previously Mapped Rat BP QTLs (95% C.l.)
S
HR
.18
Co
ng
enic
in
terv
al
Rat Chr. 18
Differentially Expressed Genes and BP QTLs on Rat Chromsome 18
28 bp insertion identified in 3' region of DE4
cDNA
Hypoxia-induced Pulmonary HypertensionHypoxia-induced Pulmonary Hypertension(Sebkhi, Wilkins, Zhao)(Sebkhi, Wilkins, Zhao)
________________________________________________________________________________________________________________________________________________________________________________________________________________________
VARWKY = 736 VARF344 = 843VARF1 = 833VARF2 = 2336
VARGENETIC = 1532Heritability = 65%
0
50
100
150
200
250
300
350
400
450
500
RV
wei
gh
t (m
g)
WKY F344 F1 F2
RV Weight QTL on Chromosome 17RV Weight QTL on Chromosome 17
1
2
3
4
5
6
7
LO
D
D1
7R
at1
3
D1
7R
at4
7
D1
7R
at4
6
D1
7R
at3
2
D1
7R
at4
3
D1
7R
at4
1
D1
7R
at1
5
D1
7R
at1
2
D1
7M
IT7
D1
7R
at6
Zhao et al 2000, Circulation
RV Weight QTL on Chromosome 17RV Weight QTL on Chromosome 17
1
2
3
4
5
6
7
LO
D
D1
7R
at1
3
D1
7R
at4
7
D1
7R
at4
6
D1
7R
at3
2
D1
7R
at4
3
D1
7R
at4
1
D1
7R
at1
5
D1
7R
at1
2
D1
7M
IT7
D1
7R
at6
Minimal interval ~ 5cMMinimal interval ~ 5cM
Congenic 1Congenic 2
WKY NTN Day 6H&E
ED1
WKY NTN Day 28H&E
WKY NormalH&E
Rat Experimental Nephrotoxic Nephritis(Cook, Duda, Smith)
LEW LewxWKYWKYxLew WKY0
25
50
75
100
Cre
sc
ents
(%
)
F2
F1
Renal histology
Chromosome 13
Lod
2
4
6
Chromosome 16
Rat NTN shows linkage to chromosomes 13 and 16
Lod
2
4
6
No. of Mendelian traits
No. of Complex Traits
Glazier, Nadeau, Aitman, 2002
Mendelian traits
All complex traits
Human complex traits
1980 1985 1990 1995 2000
Identification of Genes underlying Mendelian and Complex Traits
1980-2002
Complex Trait Genes Identified During 2003
• CTLA4, autoimmune disease in mice and humans– Ueda, Nature 423:506
• Ncf1, rat pristane-induced arthritis– Olofsson, Nature Genet 33:25
• PHF11, Human asthma and IgE– Zhang et al, Nature Genet 34:181
No. of Mendelian traits
No. of Complex Traits
Glazier, Nadeau, Aitman, 2002
Mendelian traits
All complex traits
Human complex traits
1980 1985 1990 1995 2000 2005
Identification of Genes underlying Mendelian and Complex Traits
1980-2005
Molecular lesions in genes underlying Mendelian and complex trait genes
identified to date
Missense/
nonsense
Splice Ins/del Regu-latory
Others
Mendelian *
(n=30,900)
58% 10% 31% 1% <1%
* Data from Human Gene Mutation Database, Cardiff
Missense/
nonsense
Splice Ins/del Regu-latory
Others
Mendelian *
(n=30,900)
58% 10% 31% 1% <1%
Complex
(n=27)
55% 0% 16% 23% 6%
Molecular lesions in genes underlying Mendelian and complex trait genes
identified to date
* Data from Human Gene Mutation Database, Cardiff
ACKNOWLEDGEMENTSIC/Clinical Sciences CentreAnne GlazierCaroline WallaceSaira AliKelly Sheehan-RooneyPenny NorsworthyJames Scott
NephrologyTerry CookMark DudaJenny Smith
Clinical PharmacologyKarim SebkhiMartin Wilkins
Microarray CentreHelen Causton
SUNYNada Abumrad
PragueMichal Pravenec
Vaclav Zidek
Vladimir Kren
San FranciscoTed Kurtz
SciosLarry Stanton
FundingMRC, Wellcome Trust
BHF, Affymetrix
